An improved method is described for the isolation of potato metaphase chromosomes for karyotypic and cytogenetic studies. Root tips from diploid Mexican species, Solanum pinnatisectum (2n = 2x = 24) and tetraploid cultivated S. tuberosum (2n = 4x = 48) were given four different pretreatments. The synthetic pyrethroid, Ambush, was the most stable and effective pretreatment reagent, providing the highest percentage of mitotic chromosomes at metaphase and the best spread of countable chromosomes for cytogenetic studies. Compared with an Ambush pretreatment at concentrations of 100-400 ppm, 1 to 10 ppm Ambush produced more easily distinguished chromosomes, which can be useful for comprehensive observation and karyotype analysis in both 2x and 4x potato species. This improved technique for examining mitotic chromosomes will be helpful in describing karyotypes, characterization of new hybrids, and identifying chromosome structural changes that are important in breeding schemes.